The brine shrimp nauplii synthesize Na,K-ATPase de novo. In this species, there appears to be polymorphism since two large chains (alpha1 and alpha2) can be clearly separated. The proportion of alpha1 and alpha2 changes with development so that by 40 hrs, 90 percent or more of the large chain(s) is alpha1. We will continue our studies on the site of synthesis of the nascent chains, i.e., with antibodies against alpha we will test to see whether the antibody against alpha reacts with free or membrane-bound ribosomes. Since alpha1 and alpha2 have identical peptide maps, it is likely that their primary structures are the same. We will follow post-translational processing (proteolytic or glycosylation) to see if either process is responsible for the heterogeneity. We have isolated a crude mixture of mRNA's is active in translating Na,K-ATPase. We will attempt partial purification of the Na,K-ATPase mRNA by sucrose gradient centrifugation (we expect the mRNA for Na,K-ATPase large chain to be in the 25-30 S fraction) and affinity chromatography. By standard techniques of mRNA amplification, reverse transcriptase and DNA sequencing, we should be able to determine the amino acid sequence in the alpha1, which is absolutely essential in determining the three-dimensional structure of the Na,K-ATPase.